Chromatography and Spectroscopy

Question 1

What is the chemical test and observation for the functional group alkene?

Answer 1

1. Add bromine water drop-wise

2. Bromine water decolourised from orange to colourless

Question 2

What is adsorption?

Answer 2

• Adsorption is the the process by which the solid silica holds the different substances in the mixture to its surface
• Separation is achieved by the relative adsorptions of substances with the stationary phase

Question 3

What is nuclear spin?

Answer 3

• Electrons have a property called spin and the nucleus also has a nuclear spin, that is significant if there is an ODD number of nucleons (protons and neutrons)
• Almsot all organic molecules content carbon and hydrogen, mostly as the 1H and 12C isotopes with a small proportion (1.1%) of the 13C isotope, so for the organic chemist, NMR is relevant for 1H and 13C, the isotopes with an odd number of nucleus
• NMR spectroscopy can be used to detect isotopes of the relents with odd numbers of nucleons, such as 19F and 31P, however 13C and especially 1H NMR spectroscopy are the components dorms of analysis used
• As a 1H nucleus consists of just a proton, 1H NMR is usually referred to as proton NMR

Question 4

What is stationary phase in gas chromatography?

Answer 4

• Gas chromatography is useful for separating and identifying volatile organic compounds present in a mixture
  1. The stationary phase is a solid or liquid adsorbed onto the surface of an inert solid on a long (1-30m) chromatography column is (a high boiling liquid adsorbed onto an inert solid support)
  2. Works by relative solubility, the polarity of the liquid on the column can be changed and hence changes the solubility of the the solutes in the stationary phase. This can be used to achieve better separation

Question 5

How does an NMR spectrometer work?

Answer 5

1. Radio frequency radiation has much less energy than the infrared radiation used in IR spectroscopy
2. The frequency required for resonance is proportional to the magnetic field strength and it is only in strong and uniform magnetic fields that this small quantity of energy can be detected
3. Typically a very strong super-conducting electromagnet is used, cooled to 4K by liquid helium
   - For organic chemistry, most routine spectrometer operate at radio frequencies of 100,200 or 400MHz
   - They are also found in hospitals as MRI (magnetic resonance imaging) body scanners, a technique that uses the same technology

Question 6

What is splitting like in aromatic compounds?

Answer 6

• From Figure 1, aromatic protons are expected to absorb in the range delta=6.2-8.0 ppm
• Splitting does occur but this can be difficult to interpret
• You are only expected to interpret aromatic proton as groups of protons often one or more multiplet

Question 7

What is another splitting?

Answer 7

1. In CH(CH)2 above, the two CH3 groups are in the same environment
2. Sometimes, adjacent protons may have different environments
3. In molecules such as CH2CH2CH2COOH, the central -CH2- would be split differently by the CH3 and CH2 protons
4. The resulting splitting would then be shown as a multiplet

Question 8

What is the chemical test and observation for the functional group carbonyl?

Answer 8

1. Add 2,4-dinitrophenylhydrazine

2. Orange precipitate

Question 9

What is the stationary phase in thin layer chromatography (TLC)?

Answer 9

1. Dry silica or alumina coated onto a glass or plastic surface
2. If the solid is absolutely dry, then the chromatography proceeds via adsorption. The solids are polar and therefore their speed at which the solute move up the solid depends on their polarity

Question 10

What happens in a NMR spectrometer?

Answer 10

1. In an NMR spectrometer, the sample is dissolved in a solvent and placed in a narrow NMR sample tube, together with a small amount of TMS
2. The tube is placed inside the NMR spectrometer, where it is spun to even out any imperfections in the magnetic field within the sample
3. The spectrometer is zeroed against the TMS standard ad the sample is given a pulse of radiation containing a range of radio frequencies, whilst maintaining a constant magnetic field
4. Any absorptions of energy resulting from resonance are detected and displayed on a counter screen
5. After analysis the sample can be recovered by evaporation of the solvent

Question 11

What is the mobile phase?

Answer 11

The mobile phase does move, and is normally a liquid or gas

Question 12

What are the four main types of proton that absorb over different chemical shift ranges?

Answer 12

1. H bonded to C=O or part of COOH
2. H bonded to C=C or aromatic ring
3. HC bonded to electronegative atom (e.g. O,N,Cl,Br or benze ring or C=O)
4. HC bonded to an alkyl chain
   - Factors psych as solvent, concentration, and substituents may move a peak outside of these ranges

Question 13

What are hydroxyl and amino protons like?

Answer 13

1. Organic compounds may contain protons that are not bonded to carbon atoms, for example, organic compounds often contain -OH and -NH protons
   - The functional groups involved include:
2. Alcohols, ROH, phenols ArPH, and carboxylic acids, RCOOH
3. Amides, RNH2, amides, RCONH2, and amino acids, RC(NH2)COOH
   - In solution NH and OH protons may be involved in hydrogen bonding and the the NMR peaks are often broad and of variable chemical shift
   - Example look at textbook
   - The broadening of the peaks also means that OH and NH protons are not usually involved in spin-spin coupling and all this can make assigning OH and NH protons difficult

Question 14

What is the standard reference chemical in NMR?

Answer 14

• Tetramethylsilane (TMS), (CH3)4Si, is used as the standard reference chemical against which all chemical shifts are measured
• TMS is given a chemical shift value of 0 ppm

Question 15

How do you work out the relative numbers of each type of proton?

Answer 15

1. In a carbon-13 NMR spectrum, the peak area is not directly related to the number of carbon atoms responsible for the peak, and this all change for proton NMR- the rate of the relative areas under each peak gives the ratio of the number of protons responsible for each peak
2. The NMR spectrometer measures the area under each peak as an integration trace, and the integration trace is shown wither as na extra line on the spectrum or as a printed number of the relative peaks areas and this provides invaluable information for identifying an unknown compound
   - Examples in textbooks and notes

Question 16

Describe the process of gas chromatography

Answer 16

1. A small amount of the volatile mixture is injected into the apparatus, called a gas chromatograph
2. The mobile carrier gas carries the components in the sample through the capillary column which contains the liquid stationary phase absorbed onto the solid support
3. The components slow downs they interact with the liquid stationary paste inside the column
4. The more soluble the component is in the liquid stationary phase, the slower it moves through the capillary column

Question 17

What is the chemical shift?

Answer 17

1. In an organic molecule, every carbon and hydrogen atom is bonded to other atoms, and all atoms have electrons surrounding the nucleus, which shift the energy and radio frequency needed for nuclear magnetic resonance to takes place
2. The frequency shift is measured on a scale called chemical shift, delta, in parts per million (ppm)

Question 18

What affect does the deuterium oxide have on proton exchange?

Answer 18

1. Deuterium exchanges and replaced the OH and NH protons in the sample with deuterium atoms
   - For example the following equilibrium is set up with methanol CH3OH + D2O (equilibrium arrow) CH3OD + HOD
2. So the second spectrum is essentially being run on CH3OD and as deuterium does not absorb in this chemical shift range, the OH peak disappears

Question 19

How do you analyse a gas chromatogram?

Answer 19

1. As each component of the mixture leaves the column, its relative concentration can be measured by a recorder
2. The relative amounts of each component in a mixture can be measured by the real of their respective peaks
   % of each component = (area of peak/total area of peaks) x 100%
3. Linking the column to a mass spectrometer means that as the unknown wave the column, it can be identified by comparing its fragmentation pattern with that of known compounds, and it can be identified almost instantly
4. This technique is called gas chromatography mass spectrometry (GC-MS)

Question 20

What is a stationary phase?

Answer 20

The stationary phase does not move, and is normally a solid or a liquid supported on a solid

Question 21

What is the chemical test and observation for the functional group carboxylic acid?

Answer 21

1. Add aqueous sodium carbonate

2. Effervescence

Question 22

What is proton exchange?

Answer 22

• Chemists have decided a technique called proton exchange for identifying -OH and -NH protons
  1. A proton NMR spectrum is run as normal
  2. A small volume of deuterium oxide, D2O, is added, the mixture is shaken and a second spectrum is run

Question 23

What is adsorption?

Answer 23

• Stationary Phase: A polar solid that has to be dry
• Mobile Phase: A liquid
• How does it work: The components in the mixture are usually polar compounds and are attracted to the solid phase to varying degrees. The more the solute is attracted to the solid phase, the more slowly it will move

Question 24

What is the amount of chemical shift determined by?

Answer 24

1. The amount of chemical shift is determined by chemical environment, especially the presence of nearby electronegative atoms
2. So depending on the chemical environment, NMR requires a different energy and frequency, producing absorption peaks at chemical shifts
3. This means that the carbon and hydrogen arrangement in a molecule ca be mapped out without needing to carry out conventional chemical test and without destroying the organic compound under test

Question 25

What is the chemical test and observation for the functional group aldehyde?

Answer 25

1. Add Tollens' reagent and warm | 2. Silver mirror

Question 26

What are the four main types of carbon atom that absorb over different chemical shift ranges?

Answer 26

- There are four main types of carbon atom that absorb over different chemical shift ranges 1. Carbon as part of a C=O 2. Carbon as part of C=C or aromatic ring 3. Carbon bonded to an electronegative atom e.g. O,N,Cl,Br 4. Carbon bonded to carbon - The chemical shift may also be outside of these ranges, depending on the solvent, concentration and substituents

Question 27

What is resonance?

Answer 27

1. An electron has two different spin states, and the nucleus also has two different spin states and these have different energies 2. With the right combination of a strong magnetic field and radio frequency radiation, the nucleus can absorb energy and rapidly flips between the two spin states 3. This is called resonance and the whole process gives the name 'nuclear magnetic resonance'

Question 28

What is the chemical test and observation for the functional group primary and secondary alcohols, and aldehyde?

Answer 28

1. Add acidified potassium dichromate (VI) and warm in a water bath 2. Colour change from orange to green

Question 29

What is spin-spin coupling?

Answer 29

1. A proton NMR peak can be split into sub-peaks or splitting patterns 2. These are caused by the proton's spin interacting with the spin states of nearby protons that are in different environments 3. This proves information about the number of protons bonded to adjacent carbon atoms

Question 30

What is relative solubility?

Answer 30

- Stationary Phase: a solid or a viscous liquid adsorbed onto a solid - Mobile Phase: A gas or a liquid - How does it work: The solute (in the mixture) is more soluble in one phase than another. The components are separated according to their difference solubilities in the two phases. If a solute is relatively more soluble in the mobile phase than another solute, then it will pass more quickly

Question 31

How do you determine the concentration of components in a sample?

Answer 31

- The concentrations of. a component in a sample is determined by comparing its peak integration (peak area) with values obtained from standard solutions of the component 1. Prepare standard solutions of known concentrations of the compound being investigated 2. Obtain gas chromatograms for each standard solution 3. Plot a calibration curve of peak area against concentration. This is called external calibration and offers a method for converting a peak area into a concentration 4. Obtain a gas chromatogram of the compounding investigated under the same conditions 5. Use the calibration curve to measure the concentration of the compound

Question 32

What is chromatography in general?

Answer 32

- A mixture carried by a mobile phase is moved over a stationary phase - The stationary phase tends hold back the components of the mixture to differing extents, so they move through the stationary phase at different rates and therefore separate - The components of the mixture to be separated are called solutes

Question 33

What is NMR spectroscopy?

Answer 33

1. Nuclear Magnetic Resonance (NMR) spectroscopy is used in the analysis of organic compounds 2. The technique uses a combination of a very strong magnetic field and radio frequency radiation 3. With the right combination of magnetic field strength and frequency, the nuclei of some atoms absorb this radiation 4. The energy for the absorption can be measured and recorded as an NMR spectrum

Question 34

What are retention times on a gas chromatogram?

Answer 34

1. 5 major peaks show that 5 drugs were present in the sample, identified from their retention time 2. You can see from the relative sizes of the peaks that there is a different proportion of each drug

Question 35

What is the chemical environment of a carbon atom determined by?

Answer 35

- The chemical environment of a carbon atom is determined by the position of the atom with the molecule 1. Carbon atoms that are bonded to different atoms or groups of different environment and will absorb at different chemical shifts 2. If two carbon atoms are positioned symmetrically within a molecule, they they are equivalent and have the SAME chemical shift. They will then absorb radiation at the same chemical shift and contribute to the same peak

Question 36

How is it separated in gas chromatography? What is the retention time?

Answer 36

1. The components of the mixture are separated depending on their solubility in the liquid stationary phase 2. The compounds in the mixture reach the detect at different times depending on their interactions with the stationary phase in the column 3. The compound retained in the column for the shortest time has the lowest retention time and is detected first 4. There retention time is the time taken for each comment to travel through the column

Question 37

What is chromatography used for?

Answer 37

1. Chromatography is used to separate individual components from a mixture of substances 2. All forms of chromatography have a stationary phase and a mobile phase 3. Chromatography can be used in the analysis of drugs, plastics, flavourings, air samples, and has applications in forensic science

Question 38

Where is GC used and how?

Answer 38

1. Environmental analysis: organic pollutants in water; pesticides in food 2. Forensic drug detection; minute amounts of drugs in forensic samples 3. Security operations explosives in luggage or on people 4. Space probes: the components of a planet's atmosphere

Question 39

What does carbon-13 NMR spectroscopy provide?

Answer 39

- A carbon-13 NMR spectrum provides two important pieces of information about the molecule: 1. The NUMBER of different carbon environments - from the number of peaks 2. The TYPES of carbon environment present - from the chemical shift

Question 40

What two pieces of information can be obtained from a gas chromatogram?

Answer 40

- Each component is detected as a peak on the gas chromatogram and two pieces of infuriation can be obtained from a gas chromatogram 1. Retention times can be used to identify the components present in the sample by comparing these to retention times for unknown components 2. Peak integrations (the areas under each peak) can be sued to determine the concentrations of components in the sample

Question 41

What are equivalent and non-equivalent protons?

Answer 41

- For carbon-13 NMR, you saw that the same chemical environment absorb at the same chemical shift value, these carbon atoms are EQUIVALENT to one another and for proton NMR, the same principle applies: 1. If two or more protons are EQUIVALENT, they will absorb at the sane chemical shift, increasing the size of the peak 2. Protons of different types have different chemical environments and are NON-EQUIVALENT - they absorb at different chemical shift

Question 42

What is the n+1 rule?

Answer 42

1. The splitting of a min peak into sub-peaks is called spin-spin coupling or spin-spin splitting and the number of sub peaks is one greater than the number of adjacent protons causing the splitting 2. This sounds complicated but the pattern is easy to see using the n+1 rule - For a proton with n protons attached to an adjacent carbon atom, the number of sub-peaks in a splitting pattern = n+1 3. When analysing spin-spin splitting, you are really seeing the number of hydrogen atoms on the immediately adjacent carbon atoms

Question 43

What are the disadvantages of gas chromatogram?

Answer 43

1. A mixture may have many components and each of these have to have retention times for comparison 2. Unknown compounds may have no reference times for comparison and hence identification 3. Different substances may have identical retention times 4. Substances that have high boiling points cannot be separated

Question 44

What are the the two mechanisms by which chromatography work?

Answer 44

- Relative solubility | - Adsorption

Question 45

How do you interpret a TLC plate?

Answer 45

-By comparing its Rf value with known values recorded used the same solvent system, and absorbent Rf value Rf=distance moved by the component/distance moved by the solvent front

Question 46

What does a proton NMR provide?

Answer 46

- A proton NMR spectrum provides four important pieces of information about a molecule. Firstly the spectrum provides similar information to a carbon-13 NMR spectrum but for protons: 1. The NUMBER of different proton environments - from the number of peaks 2. The TYPES of proton environments present - from the chemical shift - There are two extra pence of information: 3. The RELATIVE NUMBERS of each type of proton - form the integration traces or ratio numbers of the relative peak areas 4. The number of non-equivalent protons ADJACENT to a given proton from the spin-spin splitting pattern

Question 47

Why does spin-spin coupling occur in pairs?

Answer 47

1. In an NMR spectrum, if you see one splitting pattern there must always be another - slitting patterns occur in pair because each proton splits the signal of the other 2. This can make it very easy to spot a structural feature when analysing a molecule, as there are several very common splitting pairs that you may see in a spectrum - Look at notes

Question 48

How do gas chromatograms work 2?

Answer 48

1. A gas chromatogram is a very sensitive way of separating and identifying the components of a mixture of volatile components 2. First, pure substances are run under identical conditions and their retention times measured 3. The mixture is then run and the retention times of the components compared with those of the pure substances

Question 49

What are deuterated solvents?

Answer 49

1. Molecules of most common solvents contain carbon and hydrogen atoms, which will produce a single in both 13C and 1H NMR spectra 2. A deuterated solvent is usually used in which the 1H atoms have been replaced by 2H atoms (deuterium, D) 3. Deuterium produces no NMR single in the frequency ranges used in 1H and 13C NMR spectroscopy 4. Deuterated trichloromethane, CDCl3, is commonly used as a solvent in NMR spectroscopy, but this will still produce a peak in a carbon-13 NMR spectrum 5. The computer usually filters out this peak before displaying the spectrum

Question 50

What is the chemical test and observation for the functional group haloalkane?

Answer 50

1. Add silver nitrate and ethanol and warm to 50degrees C in a water bath 2. Chloroalkane - white precipitate Bromoalkane - cream precipitate Iodoalkane - yellow precipitate

Question 51

What is the mobile phase in gas chromatography?

Answer 51

1. The mobile phase is an inert carrier gas such as helium or neon or a volatile liquid. The mobile phase is called the carrier gas 2. The time taken for the volatile components of the mixture to move through the column is called their retention time

Question 52

What is the mobile phase in thin layer chromatography (TLC)?

Answer 52

1. A pure liquid or a mixture of liquids | 2. The chromatography depends on the relative solubilities of the solutes in the mobile and stationary phase

Question 53

Why is using a retention time beneficial?

Answer 53

- It is common to run a TLC of a sample alongside pure samples of compounds that may be present, it is then easy to identify the amino acids in the unknown sample visually, without needing to calculate any Rf values - This value means that a solute can be identified even if the chromatogram is run for different times - As long as the substances used for the stationary and mobile phases are the same, a comparison is valid

Question 54

State the property of alpha amino acids that allow them to be separated by TLC?

Answer 54

Adsorption

Question 55

How could the student analyse the chromatogram to identify the three alpha amino acids that were present?

Answer 55

1. Measure how far the each spot travels relative to the solvent front 2. Compare Rf values with that of know amino acids

Question 56

Why could the fact that several alpha amino have structures that are very similar cause problems when using TLC to analyse mixtures of alpha amino acids?

Answer 56

The amino acids have similar Rf values

Question 57

How could infrared spectroscopy be used to confirm which compound is a carboxylic acid?

Answer 57

The carboxylic acid has a peak at 2500-3300 cm^-1 for the O-H in carboxylic acids

Question 58

How would carbon 13 NMR and infrared spectroscopy allow the carboxylic acids to be identified and distinguished from one another?

Answer 58

- The carboxylic acids have a peak 2500-3300 cm^-1 for the O-H in carboxylic acid - One has 4 peaks for 4 different carbon environments - One has 3 peaks for 3 different carbon environments

Question 59

What would the difference be if the 1HNMR spectra was run but with a few drops of D20 added?

Answer 59

The peaks at 10.2-12ppm for two COOH protons would disappear as the deuterium would exchange with the COOH protons